APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2005, p. 5935–5942 Vol. 71, No. 10 0099-2240/05/$08.00ϩ0 doi:10.1128/AEM.71.10.5935–5942.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Genetic Diversity of Small in Lakes Differing by Their Trophic Status Marie Lefranc, Aure´lie The´not, Ce´cile Lepe`re, and Didier Debroas* Universite´ Blaise Pascal, Laboratoire de Biologie des Protistes UMR CNRS 6023, 63177 Aubie`re cedex, France

Received 1 December 2004/Accepted 10 May 2005

Small eukaryotes, cells with a diameter of less than 5 ␮m, are fundamental components of lacustrine planktonic systems. In this study, small- diversity was determined by sequencing cloned 18S rRNA genes in three libraries from lakes of differing trophic status in the Massif Central, France: the oligotrophic Lake Godivelle, the oligomesotrophic Lake Pavin, and the eutrophic Lake Aydat. This analysis shows that the least diversified library was in the eutrophic lake (12 operational taxonomic units [OTUs]) and the most diversified was in the oligomesotrophic lake (26 OTUs). Certain groups were present in at least two ecosystems, while the others were specific to one lake on the sampling date. Cryptophyta, Chrysophyceae, and the strictly heterotrophic eukaryotes, Ciliophora and fungi, were identified in the three libraries. Among the small eukaryotes found only in two lakes, Choanoflagellida and environmental sequences (LKM11) were not detected in the eutrophic system whereas were confined to the oligomesotrophic and eutrophic lakes. Three OTUs, linked to the , were detected only in the Aydat library, where they represented 60% of the clones of the library. Chlorophyta and Haptophyta lineages were represented by a single clone and were present only in Godivelle and Pavin, respectively. Of the 127 clones studied, classical pigmented organisms (autotrophs and mixotrophs) represented only a low proportion regardless of the library’s origin. This study shows that the small-eukaryote community composition may differ as a function of trophic status; certain lineages could be detected only in a single ecosystem.

Picoeukaryotes are probably the most abundant eukaryotes genes and have shown high phylogenetic diversity (15, 34, 39). on earth. They are found in all lakes and oceans at densities Moon-van der Staay et al. (39) identified a wide variety of from 102 to 104 cells/ml (8, 32). They constitute essential com- lineages mainly affiliated with photosynthetic classes. They re- ponents of the microbial food web and play significant roles in trieved sequences not clearly assigned to any known organisms. the geochemical cycle (5, 8, 50). The study carried out in deep Antarctic waters by Lo´pez- It is difficult to characterize these organisms by simple ob- Garcı´a et al. (34), under conditions considered inhospitable, servation with optical microscopy, and cultivation methods do showed the presence of many new lineages affiliated with non- not allow all the organisms to grow. Natural assemblages can photosynthetic groups including two new distinct be studied, without cultivation, by using chromatographic groups, which represented 65 to 76% of the clones analyzed. methods, high-performance liquid chromatography, and gas According to Moon-van der Staay et al. (39), the analysis of chromatography (4, 44). Pigment and/or fatty acid analysis can picoeukaryotic diversity in the surface waters of the Mediter- provide some information on the structure and dynamics of the ranean, North Atlantic, and Antarctic regions demonstrated phototrophic and/or heterotrophic behavior of planktonic or- the presence of many photosynthetic and heterotrophic lin- ganisms, but the phylogenetic information supplied by these eages. A large proportion of clones belonged to novel lineages methods is limited. including, novel and novel . In the last decade, the introduction of molecular techniques It should be emphasized that these studies on small aquatic into microbial ecology has greatly increased our knowledge by eukaryote diversity were conducted in marine ecosystems. identifying the smallest aquatic microorganisms and, more par- Thus, little is known about the diversity of this planktonic ticularly, prokaryotes. Within Eubacteria at least 13 novel di- community in lake systems, despite the large numbers of pig- visions have been catalogued, and certain clusters, such as mented organisms that participate in primary production (1, SAR11, appear to be significant components of the marine 51) and colorless cells that are generally considered grazers of bacterioplankton (21, 25). Novel archaeal lineages without any known cultured organisms have also been recognized (13, 20). prokaryotic and eukaryotic cells (11). These organisms are able Despite the power of molecular ecology techniques, these to use dissolved organic matter directly through the phagotro- methods have not been as widely used for microeukaryotes as phic process (49). ␮ for prokaryotes. Several recent studies have analyzed the di- In this study the diversity of small eukaryotes (0.2 to 5 m) versity of small eukaryotes (Ͻ3or5␮m), sampled in different was examined by cloning and sequencing eukaryotic rRNA oceanic ecosystems, by gene cloning and sequencing of rRNA genes in three lakes differing by their trophic status (oligotro- phic, oligomesotrophic, and eutrophic). The aim of the study was to determine (i) the structure of small eukaryotes in * Corresponding author. Mailing address: Universite´ Blaise Pascal, lacustrine systems and (ii) whether or not the composition of Laboratoire de Biologie des Protistes UMR CNRS 6023, 63177 Aubie`re cedex, France. Phone: 33 473 407837. Fax: 33 473 4077837. the small eukaryote community is dependent on the system’s E-mail: [email protected]. productivity. As far as we know, this work is the first descrip-

5935 5936 LEFRANC ET AL. APPL.ENVIRON.MICROBIOL.

TABLE 1. Main characteristics of the different lakes sampled

Chl a Maximum Sampling date Temp Oxygen concn Lake Trophic status Coordinates concn depth (m) (day/mo/yr) (°C) (mg/liter) (␮g/liter) Godivelle Oligotrophic 45°23ЈN, 2°55ЈW 55 17/07/02 14.9 18 0.02 Pavin Oligomesotrophic 45°30ЈN, 2°53ЈW 95 02/07/02 15 9.9 1.9 Aydat Eutrophic 45°39ЈN, 2°59ЈW 15 06/08/02 25.5 7.4 12.2

tion of small-eukaryote diversity in lakes by using molecular electrophoresis in a 2.5% low-melting-point agarose gel (NuSieve) at 60 mV for techniques. about 3 h. Clones from the same library (i.e., lake) that produced the same RFLP pattern were grouped together and considered members of the same operational taxonomic unit (OTU). Thereafter, the OTUs from the three libraries were MATERIALS AND METHODS checked by terminal RFLP (T-RFLP) analysis. 18S rRNA genes from clones were amplified as described above, except that the fluorescently labeled forward Study site and sampling. The study was conducted in three lakes (Massif primer 1f-FAM (6-carboxyfluorescein) (labeled at the 5Ј end with fluorescent Central, France): the oligotrophic Lake Godivelle (lac d’en haut), the oligome- sequencing dye [MWG Biotech, Germany]) was used. PCR products were puri- sotrophic Lake Pavin, and the eutrophic Lake Aydat (Table 1). The circular Lake fied using the Qiaquick PCR purification kit (QIAGEN), visualized on 1% Godivelle, situated at an altitude of 1,239 m with a maximum depth of 44 m, agarose gels, and quantified (DNA quantification kit; Sigma). Enzymatic diges- occupies a volcanic explosion crater. Lake Pavin, situated at an altitude of 1,197 tions were performed separately for each restriction enzyme used by incubating m, is a typical crater mountain lake with a maximum depth of 92 m. Aydat Lake 100 ng of PCR products with 20 U of MspI and RsaI (Sigma) at 37°C overnight. was formed when a lava flow dammed the small river Veyre. It is a dimictic lake The samples were desalted with Microcon columns (Amicon; Millipore). The with a maximum depth of 15 m, situated at an altitude of 825 m (46). Mean terminal restriction fragments (T-RFs) were separated on an automated se- chlorophyll a (Chl a) concentrations were Ͻ1, 2, and 12 ␮g/liter in Godivelle, quencer (ABI 3700), and T-RF sizes were determined using Genescan analytical Pavin and Aydat lakes, respectively. Average total phosphorus concentrations (in software. micrograms of P per liter) were 4 in Lake Godivelle, 10 in Lake Pavin, and 35 in At least one clone of each OTU was selected for sequencing. Double-stranded Lake Aydat (46). The temperatures and dissolved oxygen and Chl a concentra- plasmid DNAs from selected clones were extracted with a QIAprep Spin Mini- tions measured at the sampling date (summer 2002) are reported in Table 1. prep kit (QIAGEN). Euk-1F and an internal primer (Ek-555f [AGTCTGGTG One sample per lake was collected in the euphotic zone with a Van Dorn CCAGCAGCCGC] or Ek-NSF573 [CGCGGTAATTCCAGCTCCA]) were used bottle at the deepest point in the three lakes. Water samples (from 70 to 120 ml for partial sequencing, and a vector primer and an internal primer were used for depending on the lake) were prefiltered through 5-␮m-pore-size polycarbonate complete sequencing. Nineteen OTUs were totally sequenced. Sequencing reactions prefilters (Millipore) at a very low vacuum to prevent cell damage (pressure, Ͻ2 were performed by MWG (http//www.mwg-biotech.com). kPa) and kept in 150-ml plastic bottles for less than 2 h during transport until Phylogenetic analysis. To determine the first phylogenetic affiliation, each processing in the laboratory for microbial collection. The microbial biomass was sequence was compared with sequences available in databases using BLAST collected on 0.2-␮m-pore-size (pressure, Ͻ10 kPa) polycarbonate filters (Milli- from the National Center for Biotechnology Information and the Ribosomal pore) and stored at Ϫ80°C until nucleic acid was extracted. Database Project (2, 37). The sequences were aligned with complete sequences Nucleic acid extraction. The filters were covered with TE buffer (1ϫ Tris and Ϫ of an ARB database using the latter’s automatic alignment tool (www.arb- EDTA) and a lysosyme solution (final concentration, 250 ␮g·ml 1) and were home.de) (36). The resulting alignments were checked and corrected manually, incubated at 37°C for 30 min. Then sodium dodecyl sulfate (10%) and proteinase Ϫ considering the secondary structure of the rRNA molecule. Sequences were K (final concentration, 100 ␮g·ml 1) were added, and the filters were incubated inserted into an optimized tree according to the maximum parsimony criteria at 37°C for at least 60 min. A cetyltrimethylammonium bromide (CTAB) solu- without allowing any changes to the existing tree topology (ARB software). The tion (final concentration, 1% in a 0.7 M NaCl solution) was added, and samples resulting tree was pruned to retain the closest relatives, sequences representative were incubated at 65°C for 10 min. Nucleic acids were extracted with chloroform- of eukaryotic evolution, and our clones. The sequences were screened for po- isoamyl alcohol (24:1); the aqueous phase containing nucleic acids was kept and tential chimeric structures by using Chimera check from Ribosomal Database purified by adding phenol-chloroform-isoamyl alcohol (25:24:1). After isopropa- project II and by performing fractional treeing on the 5Ј and 3Ј ends of the nol (0.6 volume) addition, the nucleic acids were precipitated at Ϫ20°C for 12 h. sequenced DNA fragments. One obviously chimeric sequence was discarded After centrifugation, the DNA pellet was ethanol rinsed and resuspended in 50 from the analysis. ␮l of TE buffer. The DNA yield was quantified by a fluorescence assay (DNA Rarefaction analysis was performed using Analytic Rarefaction software (ver- quantification kit; Sigma), and nucleic acid extracts were stored at Ϫ20°C until sion 1.3) (www.uga.edu/ϳstrata/software/Software.html), based on the analytic analysis. solution presented by Raup (43) and Tipper (55). Eukaryotic rRNA genetic libraries. Eukaryotic 18S rRNA genes were ampli- Nucleotide sequence accession numbers. Nucleotide sequences determined in fied with eukaryote-specific primers Ek-1F (CTGGTTGATCCTTGCCAG) and this study have been deposed in GenBank under accession numbers AY642693 Ek-1520r (CYGCAGGTTCACCTAC) (33). The PCR mixture (50 ␮l) contained to AY642748. about 10 ng of environmental DNA, 200 ␮M of each deoxynucleoside triphos-

phate, 2 mM MgCl2, 10 pmol of each primer, 1.5 U of Taq DNA polymerase (Eurobio), and the PCR buffer supplied with the enzyme. Reactions were carried out in an automated thermocycler (MJ Research PTC 200-cycler) with the RESULTS AND DISCUSSION following cycle: initial denaturation at 95°C for 5 min; 30 cycles of denaturation at 95°C for 1 min, annealing at 57°C for 1 min, and extension at 72°C for 1 min, The objective of this work was to study the taxonomic com- 30 s; and a final extension at 72°C for 10 min. Several PCR products (at least four position of the community of small eukaryotes along a gradient 50-␮l samples) were pooled, precipitated with ethanol-sodium acetate, and re- of eutrophication in lacustrine environments never before de- ␮ suspended in 50 l of sterile water. scribed by molecular techniques. We analyzed three clone li- These PCR products were used to construct one clone library for each of the three lakes (Godivelle, Pavin, and Aydat) by using the TOPO TA cloning kit braries from three lakes differing in their trophic statuses: (Invitrogen) according to the manufacturer’s recommendations. oligotrophic, oligomesotrophic, and eutrophic. Fingerprint analysis and rRNA gene sequencing. Around 50 clones from each Methodological aspects. The water was prefiltered through a library were randomly picked from different plates. The presence of the 18S 5-␮m-pore-size filter to take into account the small eukaryotic rRNA gene insert in positive colonies was checked by PCR amplification using cells observable by standard epifluorescence microscopy, but flanking vector primers (M13f and M13r). Amplicons of the expected size were subsequently digested with the restriction enzyme HaeIII, and the resulting whose taxonomic characterization is often impossible, and restriction fragment length polymorphism (RFLP) products were separated by which represent a large proportion of the microorganisms in VOL. 71, 2005 GENETIC DIVERSITY OF SMALL EUKARYOTES IN LAKES 5937

the lakes (9, 52, 53). Moreover, the use of this fraction (0.2 to by 10 OTUs (A34, A43, A1, P1.35, PG5.22, A42, P34.48, 5 ␮m) makes it easier to compare the results of this study with P34.45, P34.28, and PG5.3) from three libraries. More specif- those obtained in other ecosystems (e.g., references 15 and 34), ically, sequences A1, P1.35, and A42, on the one hand, and which also used the same prefiltration. The organisms targeted sequences P34.28 and PG5.3, on the other, are associated in this way by molecular techniques correspond to small eu- with strictly heterotrophic flagellates: Paraphysomonas and karyotes with a maximum size in the region of 5 ␮m and not to Oikomonas. A43 and A34 belong to the genus Poterioochromo- the definition of picoplankton in the strict meaning of the term. nas, a mixotrophic flagellate. These three genera belong to It is well known that whatever the aquatic ecosystem, prefil- clades including phagotrophic organisms or organisms at least tration allows some cells that are larger than their nominal capable of phagotrophy (3). Clones P34.45 and PG5.22, on the pore sizes to pass through and can lead to the retention of one hand, and clone G5.2, on the other, have a different but smaller cells if the filters are clogged (10, 15). As emphasized special position among the Stramenopiles (Fig. 2). They have by Dı´ez et al. (15), the approach used to collect small eu- the strongest similarity with heterotrophic species but are not karyotes is very important. Using epifluorescence microscopy clearly associated in a clade with known organisms. The Stra- after primulin coloration (see the protocol in reference 52), we menopiles include initially heterotrophic organisms that have Ͻ observed the abundance of small eukaryotes (diameter, 5 acquired a chloroplast during their evolution (31). In our anal- ␮ m) in the nonfiltered and filtered fractions in several samples. ysis, clone G5.2 is clearly positioned in the tree before the We detected a slight decrease in total abundance (10 to 15%) acquisition of chloroplasts. Therefore, within the Strameno- but no modification of diversity inferred by morphological in- piles, we identified two sequences associated with purely het- spection. However, both water filtration and DNA amplifica- erotrophic lineages: clone G5.2, related to the Hyphochytrio- tion (58) can bias the characterization of small plankton. mycetes, and clone P34.6, included in the lineage of The diversity in the clone libraries is underestimated by and with low similarity to roenbergensis RFLP patterns generated using a single restriction enzyme (16, (88%). Among the strictly heterotrophic small eukaryotes, 47). Thus, different RFLP patterns can correspond to the same both Ciliophora and fungi were identified in the three libraries. sequences, or identical RFLP patterns can correspond to dif- Three OTUs out of six in the Ciliophora lineage have the ferent OTUs. To limit this bias, we also used T-RFLP analysis, highest similarity with the genus Oxytricha, already identified in a highly reproducible and robust technique that yields high- the marine environment in the picoplanktonic fraction by Dı´ez quality fingerprints consisting of fragments of precise sizes et al. (15). With the exception of one OTU (A44), fungi were (42). Thus, in some rare cases (three), some clones (A54 and identified in the oligotrophic and mesotrophic lakes (PG5.12, P1.31, P1.25 and PG5.34, and P34.10 and PG5.31) with the P34.43, P34.27, P1.36, G5.10, and G5.16). These OTUs were same RFLP patterns showed different T-RFLP profiles. Fur- affiliated with the lineage of Chytrids, known as parasites, for thermore, since the sequences were not similar, these were example, of green algae (35) and (6) in lacustrine considered to be different. Analysis of each library highlighted ecosystems. The fungi of these ecosystems could participate in 12 (eutrophic lake), 18 (oligotrophic lake), and 26 (oligome- regulating planktonic populations by parasitism. sotrophic lake) different OTUs. No OTUs occurred in all three Among the small eukaryotes found only in two lakes, Cho- lakes (Table 2). The diversity rarefaction curves (Fig. 1) ob- tained from the clones of Lake Aydat and, to a lesser degree, anoflagellida and environmental sequences (LKM11) were not from Lake Godivelle tend to reach a plateau, in contrast to the detected in the eutrophic system whereas Cercozoa were con- curve for Lake Pavin. More specifically, with the clone library fined to the oligomesotrophic and eutrophic lakes. Among from Lake Aydat, the curve clearly shows saturation of diver- these lineages, Cercozoa have the highest diversity (six OTUs), sity, allowing us to deduce that, in this case, this library is and with the exception of one sequence, these OTUs are not Յ certainly representative of the composition of small eukaryotic closely related to any in the database ( 88%) (Table 2). The plankton for the period studied, whereas the library of Lake Cercozoa are a complex group of eukaryotes, encompassing a Pavin represents only the most abundant clones. Thus, the wide variety of organisms and including some of the most highest diversity was observed in the oligomesotrophic lake abundant nonphotosynthetic amoebae, flagellates, and plas- (Lake Pavin). modiophorid pathogens known. The morphological, eco- More exactly, certain groups were present in at least two logical, and genetic diversity of the Cercozoa is enormous (27), ecosystems, while the others were specific to one lake on the and they are present in many different environments (47). In sampling date (Table 2; Fig. 2). our libraries, five OTUs out of six were affiliated with the genus Lineages present in at least two lakes. Cryptophyta were Cercomonas, with a low similarity on average (87%). These represented by OTUs from the three libraries that mostly cells, which are extremely common in almost all types of soil (4 stemmed from the oligotrophic (three OTUs) and oligome- to 10 ␮m), are highly metabolic with strong amoeboid proper- sotrophic (seven OTUs) ecosystems. These OTUs formed ties. The second cercozoan genus is affiliated with Heteromita three clearly distinct clades (G5.11, P1.27, P1.30, and P34.3; globosa (one OTU in the Lake Pavin library), whose globular A54, P1.31, P1.25, and PG5.34; P34.10 and PG5.31) (Fig. 2). cells are approximately 4 to 6 ␮m long (18). The clade LKM11, The presence of this lineage in the smallest planktonic fraction grouping sequences G5.3, P34.42, PG5.28, and P34.11, is agrees with previous results obtained from lacustrine ecosys- clearly differentiated on the tree, while at the same time it is tems (26, 49) and from lakes studied in the geographic area associated with fungi. These sequences, first defined in the where Cryptophyta predominate among the pigmented organ- study by Van Hannen et al. (56), are affiliated with nonculti- isms (9, 52, 53). Chrysophyceae, which include autotrophic, vable eukaryotes taken from a lacustrine ecosystem. These mixotrophic, and heterotrophic taxa (3, 23), were represented organisms, associated with the decomposition of algae and 5938 LEFRANC ET AL. APPL.ENVIRON.MICROBIOL.

TABLE 2. Number of clones belonging to each OTU in genetic libraries and phylogenetic affiliations of the representative clones sequenced

Similarity No. of clones in library of Lake: Taxon OTU Closest relative (%) Godivelle Pavin Aydat Chlorophyta PG5.14 Mychonastes homosphaera 99 1

Haptophyceae P34.19 Chrysochromulina throndsenii 97 2

Cryptophyta P34.10 Storeatula major 89 5 PG5.31 Storeatula major 88 1 P1.25 Chroomonas sp. 89 1 PG5.34 Chroomonas sp. 86 1 P1.31 Chroomonas sp. 88 1 A54 Chroomonas sp. 89 3 G5.11 Geminigera cryophila 98 1 P1.30 Geminigera cryophila 98 1 P34.3 Geminigera cryophila 98 1 P1.27 Geminigera cryophila 96 1 P1.40 Geminigera cryophila sp2 95 1

Chrysophyceae P34.48 Hibberdia magna 94 1 A34 Poterioochroomonas malhamensis 94 1 A43 Poterioochroomonas malhamensis 97 2 P34.28 Oikomonas mutabilis 92 1 PG5.3 Oikomonas mutabilis 91 1 A1 Paraphysomonas butcheri 98 1 A42 Paraphysomonas 97 4 P1.35 Paraphysomonas bandaiensis 97 1 PG5.22 Paraphysomonas foraminifera 96 2 P34.45 Spumella elongata 95 1

Bicosoecida P34.6 Cafeteria roenbergensis 88 1

Hyphochytriomycetes G5.2 Rhizidiomyces apophysatus 92 1

Dinophyceae G5.1 Prorocentrum mexicanum 94 2 PG5.8 Prorocentrum mexicanum 93 3 G5.7 Gymnodinium beii 93 1

Ciliophora P34.38 Glaucoma chattoni 97 1 PG5.26 Oxytricha nova 91 4 PG5.20 Oxytricha nova 90 1 A27 Oxytricha nova 91 1 P34.44 Prorodon teres 81 1 P1.24 Prorodon teres 90 7

Perkinsozoa A20 Perkinsus marinus 87 18 A48 Perkinsus marinus 86 9 A31 Perkinsus marinus 90 1

Cercozoa P1.23 Cercomonas sp. 86 2 A50 Cercomonas sp. 86 4 P1.18 Cercomonas sp. 95 1 P34.13 Cercomonas sp. 87 1 A51 Cercomonas sp. 79 1 P34.14 Heteromita globosa 88 1

Choanoflagellida P1.39 Diaphanoeca grandis 89 1 PG5.16 Diaphanoeca grandis 90 1

Fungi PG5.12 Spizellomyces acuminatus 93 1 A44 Spizellomyces acuminatus 90 2 P34.27 Spizellomyces acuminatus 92 1 P1.36 Spizellomyces acuminatus 90 1 G5.10 Spizellomyces acuminatus 87 1 G5.16 Spizellomyces acuminatus 83 13 P34.43 Spizellomyces acuminatus 93 1

Environmental sequences P34.42 Unidentified eukaryote LKM11 92 1 P34.11 Unidentified eukaryote LKM11 87 2 PG5.28 Unidentified eukaryote LKM11 84 5 G5.3 Unidentified eukaryote LKM11 85 1 VOL. 71, 2005 GENETIC DIVERSITY OF SMALL EUKARYOTES IN LAKES 5939

FIG. 1. Rarefaction curves determined for the different RFLP patterns of 18S rRNA gene clones. The number of different RFLP patterns was determined after digestion with the restriction endonuclease HaeIII.

cyanobacteria (56), could participate in the decomposition of This strong proportion of heterotrophs is in agreement with detritus in the oligotrophic and oligomesotrophic systems. the results of counts by epifluorescence microscopy, which Lineages specific to one lake. Some taxa seem to be specific show that the pigmented organisms generally account for only to the ecosystem studied. Thus, three OTUs, linked to the new a low proportion of small eukaryotes in lakes of this area (9, 52, phylum of Perkinsozoa, which includes the marine parasites 53). It may be noted that Chlorophyta are represented by only Perkinsus and Parvilucifera (41), were detected only in the one sequence, of which there is only one example in the Godi- Aydat library (A31, A48, and A20). They represent about 60% velle library, whereas this lineage is reputed to be widespread of the Aydat library and are affiliated with low similarities (86 in marine and lacustrine ecosystems (26). Fatty acid analysis to 90%) to Perkinsus marinus. This result raises the possibility performed for the 0.2- to 5-␮m fraction of Lakes Godivelle and that the small eukaryotes of this lake played an important role Pavin shows that Chlorophyceae may be present in the oligo- in controlling algal populations during the study period. trophic lake (unpublished data). The sampling period, as well Among the pigmented organisms, the Haptophyta, Dinoflagel- as DNA extraction and amplification problems specific to this lata, and Chlorophyta lineages were found only in one ecosys- lineage, could explain their low presence. Thus, the use of bead tem. The sequence associated with Haptophyta is strongly beating or freeze-thaw cycles (48) could improve DNA extrac- affiliated with the genus Chrysochromulina (Table 2), a tion. However, these methodological considerations are prob- phagotrophic phytoflagellate (29). Three OTUs (G5.7, PG5.8, ably insufficient for explaining the results obtained in this and G5.1) from the Godivelle library (oligotrophic) are affili- study; studies conducted in the marine environment using sim- ated with the Dinoflagellata, which include heterotrophic and ilar extraction and amplification techniques have detected autotrophic taxa, whose evolution may be linked to tertiary many sequences affiliated with green algae (15, 39). endosymbiosis (28), and more particularly with the autotrophic Some lineages were present in all ecosystems. However, this flagellates Gymnodiniales and Prorocentrales. Their detection study shows that the diversity of small-eukaryote communities in this planktonic fraction may be due to filtration problems or varies from lake to lake, with some lineages being present only else to the existence of unidentified small Dinoflagellata. The in a given lake and absent from others. For example, Perkin- low levels of similarity calculated (93 and 94%) with known sozoa were found only in the Aydat library, while Cercozoa Dinoflagellata and the fact that these flagellates have already were never detected in Lake Godivelle. Another factor that been identified in the picoplanktonic fraction of marine eco- has to be taken into account is the relative importance of systems by the same molecular techniques (15, 39) could con- clones. Thus, the Godivelle library was dominated by fungi firm the presence in this smallest fraction of a potentially new (31% of the clones) and the Aydat library by Perkinsozoa genotype belonging to this lineage. (60%). It is difficult to characterize these organisms by simple Small-eukaryote diversity in relation to trophic status. The observation with optical microscopy, and these variations are phylogenetic position of a clone enables hypotheses as to not always detectable. For example, Poterioochromonas, which whether an organism is pigmented or colorless to be made is a flagellate with loricae, is often difficult to identify because (Table 3) (3, 14, 15, 29). For all three lacustrine libraries, we the loricae are inconspicuous when examined by light micros- observed that 30 clones were considered to be pigmented (au- copy. Cells can also escape from their loricae and are then totrophic or mixotrophic) whereas 94 were affiliated with het- indistinguishable from Ochromonas spp. (14, 17). Paraphy- erotrophic lineages (Table 3) (the 2 OTUs P34.45 and PG5.22 somonas is characterized by siliceous scales on the cell surface, belonging to Stramenopiles have been classified as indetermi- but these scales cannot be easily seen with light microscopy (7). nate). The clones classified as heterotrophic accounted for Thus, several taxa identified here have certain morphological more than 60% of the clones studied, whatever the library. similarities and were probably not identified. However, as in 5940 LEFRANC ET AL. APPL.ENVIRON.MICROBIOL.

FIG. 2. Phylogenetic tree of eukaryotic small-subunit rRNA genes showing the positions of environmental clones. The tree was constructed using the ARB software package. VOL. 71, 2005 GENETIC DIVERSITY OF SMALL EUKARYOTES IN LAKES 5941

TABLE 3. Diversity of pigmented versus nonpigmented small eukaryotes could be limited by nutrient resources in an oligo- eukaryotes in clone libraries from lakes differing in trophic environment and by predation and competition in a trophic status eutrophic environment. As in marine environments (57), the No. of OTUs (clones) in Lake: diversity of heterotrophic organisms (35 OTUs) may be higher Taxon Godivelle Pavin Aydat than that of autotrophic organisms (19 OTUs) in the smallest (oligotrophic) (oligomesotrophic) (eutrophic) planktonic fraction (Table 3). More specifically, on studying the libraries independently, it can be seen that the ratio be- Overall total 18 (41) 26 (39) 12 (47) tween colorless OTUs and pigmented OTUs increased with Pigmented the trophic status and was highest in the eutrophic ecosystem. Chlorophyta 1 0 0 However, the latter ecosystem differs from the other two by its Haptophyceae 0 1 0 low diversity and a library strongly dominated by Perkinsozoa. Cryptophyta 3 7 1 Chrysophyceae 0 1 2 To a certain extent, this trend appears to be of the same type Dinoflagellata 3 0 0 as that described by Vaulot et al. (57), who showed that this Total 7 (10) 9 (14) 3 (6) ratio is higher in coastal than in oceanic environments. Generally, this study showed that the clones in these ecosys- Colorless tems tend to form specific clades, even when related to a Fungi 3 3 1 Environmental samples 2 2 0 clearly defined phylogenetic group; moreover, many similari- Choanoflagellates 1 1 0 ties remain lower than 90%. This undoubtedly results from the Cercozoa 0 4 2 fact that despite their ecological importance, few studies have Chrysophyceae 1 2 2 dealt with the small eukaryotes or picoeukaryotes of aquatic Bicosoecida 0 1 0 1 0 0 ecosystems, and thus, few 18S rRNA gene sequences are avail- Ciliophora 2 3 1 able. The few studies that do exist concern marine pelagic Perkinsozoa 0 0 3 environments and very specific marine environments such as Total 10 (29) 16 (24) 9 (41) hydrothermal sediments (33). The clones in this study are not affiliated with the new Alveolata (groups I and II) (40) or Indeterminate Stramenopiles 1 1 0 Stramenopiles (15) lineages. Chrysophyceae appear to domi- Total 1 (2) 1 (1) 0 (0) nate in this latter clade, and they are an essential and ubiqui- tous component of plankton in freshwater environments. How- ever they are rarely dominant in marine environments. It should also be noted that in this study, in contrast to studies in most studies conducted in the marine environment using clon- marine environments, diatoms were absent from this plank- ing-sequencing techniques (15, 34, 38, 39, 60), these data were tonic fraction (0.2 to 5 ␮m). These results are in agreement obtained from a single sample collected from one point on a with those obtained by microscopy counts, which very rarely single date, and therefore seasonal variations (9, 52, 53) are detect any small diatoms such as Cyclotella in the lakes stud- not taken into account. Moreover, according to Finlay (19), ied. Clones belonging to fungi also accounted for a consider- free-living microbial eukaryotes, such as , are probably able proportion, but these have not been detected or represent sufficiently abundant to have a worldwide distribution. This only a low percentage of the marine clone libraries (15, 34). view, for example, expects that microbial eukaryotes such as This study shows that as with other planktonic communities, Ascomycetes will prove to be nearly ubiquitous geographically, the small eukaryotes of lacustrine ecosystems described by whereas the distribution of these organisms tends to show molecular techniques tend to show some differences from some geographical differentiation (22). On the other hand, those of oceanic systems and vary as a function of trophic level. recent reports demonstrate that limited dispersal is also pos- On the other hand, phylogenetic determination highlights the sible for some prokaryotes (59). Finally, according to Coleman presence of original clades such as Perkinsozoa in the eutro- (12), Finlay’s assumption may be acceptable only for marine phic lake or chytrids in the two other lakes. The abundance of protistans and it would be less likely to apply to freshwater potentially parasitic organisms probably plays a significant role microbial eukaryotes. Many studies have also shown that the in controlling the population dynamics of in aquatic population composition of free-living microbial eukaryotes, systems. However, their quantitative and functional impor- such as microalgae and ciliates, varies in relation to the trophic tance remains to be determined. status (29, 45). It is therefore possible that the variations in REFERENCES lacustrine small-eukaryote community composition can be par- 1. Agawin, N. S. R., C. M. Duarte, and S. Agustı´. 2000. Nutriment and tem- tially explained by this factor. The OTU distribution (Fig. 1 perature control of the contribution of picoplankton to phytoplankton bio- and Table 3) may be related to the distributions determined in mass and production. Limnol. Oceanogr. 45:591–600. aquatic environments for algae, the bacteria belonging to the 2. Altschul, S. F., T. L. Madden, A. A. Scha¨ffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSIBLAST: a new generation Cytophaga-Flavobacterium-Bacteroides group (24), and other of protein database search programs. Nucleic Acids Res. 25:3389–3402. organisms. Thus, it was possible to demonstrate under exper- 3. Andersen, R. A., Y. Van de Peer, D. Potter, J. P. Sexton, M. Kawachic, and imental conditions that algal diversity follows a hump-shaped T. LaJeunesse. 1999. Phylogenetic analysis of the SSU rRNA from members of the Chrysophyceae. 150:71–84. progression along a eutrophication gradient and that this pro- 4. Ansotegui, A., A. Sarobe, J. M. Trigueros, I. Urrutxurtu, and E. Orive. 2003. gression results from a compromise between competition, pre- Size distribution of algal pigments and phytoplankton assemblages in a coastal-estuarine environment: contribution of small eukaryotic algae. J. dation, accessibility of nutrient resources, and many other eco- Plankton Res. 25:341–355. logical processes (30, 54). The development of small 5. Azam, F., T. Fenchel, J. G. Field, J. S. Gray, L. A. Meyer, and F. Thingstad. 5942 LEFRANC ET AL. APPL.ENVIRON.MICROBIOL.

1983. The ecological role of water column microbes in the sea. Mar. Ecol. 2001. Unexpected diversity of small eukaryotes in deep-sea Antarctic plank- Prog. Ser. 10:257–263. ton. Nature 409:603–607. 6. Canter, H. M., and G. H. Jaworski. 1981. The effect of light and darkness 35. Lopez-Llorca, L. V., and P. Hernandez. 1996. Infection of the green alga upon infection of formosa Hassall by the chytrid Rhizophydium Oocystis lacustris Chod with the Chytrid Diplochytridium deltanum planktonicum Canter emend. Ann. Bot. 47:13–30. (Masters) Karling. An SEM study. Micron 27:355–358. 7. Caron, D. A., R. J. Gast, E. L. Lim, and M. R. Dennet. 1999. Protistan 36. Ludwig, W., O. Strunk, R. Westram, L. Richter, H. Meier, Yadhukumar, A. community structure: molecular approaches for answering ecological ques- Buchner, T. Lai, S. Steppi, G. Jobb, W. Fo¨rster, I. Brettske, S. Gerber, A. W. tions. Hydrobiologia 401:215–227. Ginhart, O. Gross, S. Grumann, S. Hermann, R. Jost, A. Ko¨nig, T. Liss, R. 8. Caron, D. A., E. R. Peele, E. L. Lim, and M. R. Dennett. 1999. Picoplankton Lu¨ßmann, M. May, B. Nonhoff, B. Reichel, R. Strehlow, A. Stamatakis, N. and nanoplankton and their trophic coupling in the surface waters of the Stuckmann, A. Vilbig, M. Lenke, T. Ludwig, A. Bode, and K.-H. Schleifer. Sargasso Sea south of Bermuda. Limnol. Oceanogr. 44:259–272. 2004. ARB: a software environment for sequence data. Nucleic Acids Res. 9. Carrias, J. F., C. Amblard, and G. Bourdier. 1996. Protistan bacterivory in an 32:1363–1371. oligomesotrophic lake: importance of attached ciliates and flagellates. Mi- 37. Maidak, B. L., G. J. Olsen, N. Larsen, R. Overbeek, M. J. McCaughey, and crob. Ecol. 31:249–268. C. R. Woese. 1997. The RDP (ribosomal database project). Nucleic Acids 10. Carrias, J. F., A. Thouvenot, C. Amblard, and T. Sime-Ngando. 2001. Dy- Res. 25:109–111. namics and growth estimates of planktonic protists during spring in Lake 38. Massana, R., L. Guillou, B. Dı´ez, and C. Pedros-Alio. 2002. Unveiling the Pavin, France. Aquat. Microb. Ecol. 24:163–174. organisms behind novel eukaryotic ribosomal DNA sequences from the 11. Carrick, H. J., G. L. Fahnenstiel, E. F. Stoermer, and R. G. Wetzel. 1991. ocean. Appl. Environ. Microbiol. 68:4554–4558. Observations on the importance of the zooplankton-protozoan trophic link. 39. Moon-van der Staay, S. Y., R. De Wachter, and D. Vaulot. 2001. Oceanic 18S Limnol. Oceanogr. 36:1335–1345. rDNA sequences from picoplankton reveal unsuspected eukaryotic diversity. 12. Coleman, A. W. 2000., The significance of a coincidence between evolution- Nature 409:607–610. ary landmarks found in mating affinity and a DNA sequence. Protist 151:1–9. 40. Moreira, D., and P. Lo´pez-Garcı´a. 2002. The molecular ecology of microbial 13. Delong, E. F. 1992. Archaea in coastal marine environments. Proc. Natl. eukaryotes unveils a hidden world. Trends Microbiol. 10:31–38. Acad. Sci. USA 89:5685–5689. 41. Noren, F., O. Moestrup, and A. S. Rehnstam-Holm. 1999. Parvilucifera infectans 14. De Puytorac, P., J. Grain, and J. P. Mignot. 1987. Pre´cis de Protistologie. Noren et Moestrup gen. et sp. nov. (Perkinsozoa phylum nov.): a parasitic Editions Boube´e and Fondation Polignac, Paris, France. flagellate capable of killing toxic microalgae. Eur. J. Protistol. 35:233–254. 15. Dı´ez, B., C. Pedro´s-Alio´, and R. Massana. 2001. Study of genetic diversity of 42. Osborn, A. M., E. R. B. Moore, and K. N. Timmis. 2000. An evaluation of eukaryotic picoplankton in different oceanic regions by small-subunit rRNA terminal-restriction fragment length polymorphism (T-RFLP) analysis for gene cloning and sequencing. Appl. Environ. Microbiol. 67:2932–2941. the study of microbial community structure and dynamics. Environ. Micro- 16. Dunbar, J., S. Takala, S. M. Barns, J. A. Davis, and C. R. Kuske. 1999. Levels biol. 2:39–50. of bacterial community diversity in four arid soils compared by cultivation and 43. Raup, D. M. 1975. Taxonomic diversity estimation using rarefaction. Paleo- 16S rDNA gene cloning. Appl. Environ. Microbiol. 65:1662–1669. biology 1:333–342. 17. Edgar, S. M., and R. A. Andersen. 2003. The systematic of Ochromonas 44. Reuss, N., and L. K. Poulsen. 2002. Evaluation of fatty acids as biomarkers (Chrysophyceae). J. Phycol. 39:14–19. for a natural plankton community. A field study of a spring bloom and a 18. Ekelund, F., N. Daugbjerg, and L. Fredslund. 2004. Phylogeny of Heteromita, post-bloom period off West Greenland. Mar. Biol. 141:423–434. Cercomonas and Thaumatomonas based on SSU rDNA sequences, including 45. Reynols, C. S. 1984. Phytoplankton periodicity: the interaction of form, the description of Neocercomonas jutlandica sp. nov., gen. nov. Eur. J. Pro- function and environmental variability. Freshwat. Biol. 14:111–142. tistol. 40:119–135. 46. Rioual, P. 2002. Limnological characteristics of 25 lakes of the French Massif 19. Finlay, B. J. 2002. Global dispersal of free-living microbial eukaryote species. Central. Ann. Limnol. 38:311–327. Science 296:1061–1063. 47. Romari, K., and D. Vaulot. 2004. Composition and temporal variability of 20. Fuhrmann, J. A., K. MacKallum, and A. A. Davis. 1992. Novel major ar- picoeukaryote communities at a coastal site of the English Channel from 18S chaebacterial group from marine plankton. Nature 356:148–149. rDNA sequences. Limnol. Oceanogr. 49:784–798. 21. Giovanonni, S. J., T. B. Britschgi, C. L. Moyer, and K. G. Field. 1990. 48. Savin, M. C., J. L. Martin, M. Legresley, M. Giewat, and J. Rooney-Varga. Genetic diversity in Sargasso sea bacterioplankton. Nature 345:60–63. 2004. Plankton diversity in the Bay of Fundy as measured by morphological 22. Green, J. L., A. J. Holmes., M. Westoby, I. Oliver, D. Briscoe, M. Danger- and molecular methods. Microb. Ecol. 48:51–65. field, M. Gillings, and A. J. Beattie. 2004. Spatial scaling of microbial eu- Sherr, E. B. karyote diversity. Nature 432:747–750. 49. 1988. Direct use of high molecular weight polysaccharides by 335: 23. Hahn, M. W., and M. G. Ho¨fle. 2001. Grazing of and its effect on heterotrophic flagellates. Nature 1225–1227. populations of aquatic bacteria. FEMS Microbiol. Ecol. 35:113–121. 50. Stockner, J. G., and N. J. Antia. 1986. Algal picoplankton from marine and 24. Horner-Devine, M. C., M. A. Leibold, V. H. Smith, and B. J. M. Bohannan. freshwater ecosystems: a multidisciplinary perspective. Can. J. Fish. Aquat. 2003. Bacterial diversity patterns along a gradient of primary productivity. Sci. 43:2472–2503. Ecol. Lett. 6:613–622. 51. Stockner, J. G., and K. S. Shortreed. 1989. Algal picoplankton production 25. Hugenholtz, P., B. M. Goebel, and N. R. Pace. 1998. Impact of culture- and contribution to food-webs in oligotrophic British Columbia lakes. Hy- independent studies on the emerging phylogenetic view of bacterial diversity. drobiologia 173:151–166. J. Bacteriol. 180:4765–4774. 52. Thouvenot, A., D. Debroas, M. Richardot, L. B. Jugnia, and J. De´vaux. 2000. 26. Johnson, P. W., and J. M. Sieburth. 1982. In situ morphology and occurrence A study of changes between years in the structure of plankton community in of eucaryotic phototrophs of bacterial size in the picoplancton of estuarine a newly-flooded reservoir. Arch. Hydrobiol. 149:131–152. and oceanic waters. J. Phycol. 8:312–327. 53. Thouvenot, A., M. Richardot, D. Debroas, and J. De´vaux. 1999. Bacterivory 27. Keeling, P. J. 2001.; Foraminifera and Cercozoa are related in actin phylog- of metazooplankton, ciliates and flagellates in a newly flooded reservoir. J. eny: two orphans find a home? Mol. Biol. Evol. 18:1551–1557. Plankton Res. 21:1659–1679. 28. Kuvardina, O. N., B. S. Leander, V. V. Aleshin, A. P. Myl’nikov, P. J. 54. Tilman, D. 1982. Resource competition and community structure. Princeton Keeling, and T. G. Simdyanov. 2002. The phylogeny of colpodellids (Eu- University Press, Princeton, N.J. karyota, Alveolata) using small subunit rRNA genes suggests they are the 55. Tipper, J. C. 1979. Rarefaction and rarefiction—the use and abuse of a free-living ancestors of apicomplexans. J. Eukaryot. Microbiol. 49:498–504. method in paleontology. Paleobiology 5:423–434. 29. Laybourn-Parry, J. 1992. Protozoan plankton ecology. Chapman and Hall, 56. Van Hannen, E. J., W. Mooij, M. P. van Agterveld, H. J. Gons, and H. J. London, United . Laanbroek. 1999. Detritus-dependent development of the microbial com- 30. Leibold, M. A. 1999. Biodiversity and nutrient enrichment in pond plank- munity in an experimental system: qualitative analysis by denaturing gradient tonic communities. Evol. Ecol. Res. 1:73–95. gel electrophoresis. Appl. Environ. Microbiol. 65:2478–2484. 31. Leipe, D. D., S. M. Tong, C. L. Goggin, S. B. Slemenda, N. J. Pieniazek, and 57. Vaulot, D., K. Romari, and F. Not. 2002. Are autotrophs less diverse than M. L. Sogin. 1996. 16S-like rDNA sequences from Developayella elegans, heterotrophs in marine picoplankton? Trends Microbiol. 10:266–267. Labyrinthuloides haliotidis, and lacertae confirm that the stra- 58. Von Wintzingerode, F., U. B. Goebel, and E. Stackebrandt. 1997. Determi- menopiles are a primarily heterotrophic group. Eur. J. Protistol. 32:449–458. nation of microbial diversity in environmental samples: pitfalls of PCR-based 32. Li, W. K. W., D. V. Subba Rao, W. G. Harrison, J. C. Smith, J. J. Cullen, B. rRNA analysis. FEMS Microbiol. Rev. 21:213–229. Irwin, and T. Platt. 1994. Autotrophic picoplankton in the tropical ocean. 59. Whitaker, R. I., D. W. Grogan, and J. W. Taylor. 2003. Geographic barriers Science 219:292–295. isolate endemic populations of hyperthermophilic archaea. Science 301:976– 33. Lo´pez-Garcı´a, P., H. Philippe, F. Galle, and D. Moreira. 2003. Autochthonous 978. eukaryotic diversity in hydrothermal sediment and experimental microcoloniz- 60. Yuan, J., M. Chen, S. Peng, H. Zhou, Y. Chen, and L. Qu. 2004. Genetic ers at the Mid-Atlantic Ridge. Proc. Natl. Acad. Sci. USA 100:697–702. diversity of small eukaryotes from the coastal waters of Nansha islands in 34. Lo´pez-Garcı´a, P., F. Rodrı´guez-Valera, C. Pedro´s-Alio´, and D. Moreira. China. FEMS Microbiol. Lett. 240:163–170.